Anthocyanins and their physiologically relevant metabolites alter the expression of IL-6 and VCAM-1 in CD40L and oxidized LDL challenged vascular endothelial cells.

SCOPE: In vitro and in vivo studies suggest that dietary anthocyanins modulate cardiovascular disease risk; however, given anthocyanins extensive metabolism, it is likely that their degradation products and conjugated metabolites are responsible for this reported bioactivity. METHODS AND RESULTS: Human vascular endothelial cells were stimulated with either oxidized LDL (oxLDL) or cluster of differentiation 40 ligand (CD40L) and cotreated with cyanidin-3-glucoside and 11 of its recently identified metabolites, at 0.1, 1, and 10 µM concentrations. Protein and gene expression of IL-6 and VCAM-1 was quantified by ELISA and RT-qPCR. In oxLDL-stimulated cells the parent anthocyanin had no effect on IL-6 production, whereas numerous anthocyanin metabolites significantly reduced IL-6 protein levels; phase II conjugates of protocatechuic acid produced the greatest effects (>75% reduction, p ≤ 0.05). In CD40L-stimulated cells the anthocyanin and its phase II metabolites reduced IL-6 protein production, where protocatechuic acid-4-sulfate induced the greatest reduction (>96% reduction, p ≤ 0.03). Similarly, the anthocyanin and its metabolites reduced VCAM-1 protein production, with ferulic acid producing the greatest effect (>65% reduction, p ≤ 0.04). CONCLUSION: These novel data provide evidence to suggest that anthocyanin metabolites are bioactive at physiologically relevant concentrations and have the potential to modulate cardiovascular disease progression by altering the expression of inflammatory mediators.

Methods for isolating, identifying, and quantifying anthocyanin metabolites in clinical samples.

The metabolic fate of anthocyanins until recently was relatively unknown, primarily as a result of their instability at physiological pH and a lack of published methods for isolating and identifying their metabolites from biological samples. The aim of the present work was to establish methods for the extraction and quantification of anthocyanin metabolites present in urine, serum, and fecal samples. 35 commercial and 10 synthetic analytes, including both known and predicted human and microbial metabolites of anthocyanins, were obtained as reference standards. HPLC and MS/MS conditions were optimized for organic modifier, ionic modifier, mobile phase gradient, flow rate, column type, MS source, and compound dependent parameters. The impact of sorbent, solvent, acid, preservative, elution, and evaporation on solid phase extraction (SPE) efficiency was also explored. The HPLC-MS/MS method validation demonstrated acceptable linearity (R(2), 0.997 ± 0.002) and sensitivity (limits of detection (LODs): urine, 100 ± 375 nM; serum, 104 ± 358 nM; feces 138 ± 344 nM), and the final SPE methods provided recoveries of 88.3 ± 17.8% for urine, 86.5 ± 11.1% for serum, and 80.6 ± 20.9% for feces. The final methods were applied to clinical samples derived from an anthocyanin intervention study, where 36 of the 45 modeled metabolites were detected within urine, plasma, or fecal samples. The described methods provide suitable versatility for the identification and quantification of an extensive series of anthocyanin metabolites for use in future clinical studies exploring absorption, distribution, metabolism, and elimination.

Synthesis and biological evaluation of nitric oxide-donating analogues of sulindac for prostate cancer treatment.

A series of analogues of the non-steroidal anti-inflammatory drug (NSAID) sulindac 1 were synthesised tethered to nitric oxide (NO) donating functional groups. Sulindac shows antiproliterative effects against immortal PC3 cell lines. It was previously demonstrated that the effect can be enhanced when tethered to NO releasing groups such as nitrate esters, furoxans and sydnonimines. To explore this approach further, a total of fifty-six sulindac-NO analogues were prepared and they were evaluated as NO-releasing cytotoxic agents against prostate cancer (PCa) cell lines. Compounds 1k and 1n exhibited significant cytotoxic with IC50 values of 6.1±4.1 and 12.1±3.2µM, respectively, coupled with observed nitric oxide release.

Development and characterization of glutamyl-protected N-hydroxyguanidines as reno-active nitric oxide donor drugs with therapeutic potential in acute renal failure.

Acute renal failure (ARF) has high mortality and no effective treatment. Nitric oxide (NO) delivery represents a credible means of preventing the damaging effects of vasoconstriction, central to ARF, but design of drugs with the necessary renoselectivity is challenging. Here, we developed N-hydroxyguanidine NO donor drugs that were protected against spontaneous NO release by linkage to glutamyl adducts that could be cleaved by γ-glutamyl transpeptidase (γ-GT), found predominantly in renal tissue. Parent NO donor drug activity was optimized in advance of glutamyl adduct prodrug design. A lead compound that was a suitable substrate for γ-GT-mediated deprotection was identified. Metabolism of this prodrug to the active parent compound was confirmed in rat kidney homogenates, and the prodrug was shown to be an active vasodilator in rat isolated perfused kidneys (EC50 ~50 µM). The data confirm that glutamate protection of N-hydroxyguanidines is an approach that might hold promise in ARF.

Novel amino acids: synthesis of furoxan and sydnonimine containing amino acids and peptides as potential nitric oxide releasing motifs.

The incorporation of furoxan and sydnonimine ring systems into amino acid side chains is demonstrated with the preparation of four novel amino acids which carry these nitric oxide-releasing motifs. N-((4-Nitrophenoxy)carbonyl)-3-phenylsydnonimine 9 and bis(phenylsulfonyl)furoxan 10 are the key intermediates for introducing the heterocycle side chains onto appropriate amine and alcohol functionalities respectively. Furoxan 5 and 7 both displayed NO release based on determination of nitrite production. Orthogonal amino acid protecting group strategies were deployed to demonstrate that the amino acids could be incorporated into peptide frameworks. By way of demonstration the amino acids were placed centrally into several tripeptide motifs. Griess test assays showed that these amino acids released NO in the presence of γ-glutathione (GST).

Human metabolism and elimination of the anthocyanin, cyanidin-3-glucoside: a (13)C-tracer study.

BACKGROUND: Evidence suggests that the consumption of anthocyanin-rich foods beneficially affects cardiovascular health; however, the absorption, distribution, metabolism, and elimination (ADME) of anthocyanin-rich foods are relatively unknown. OBJECTIVE: We investigated the ADME of a (13)C5-labeled anthocyanin in humans. DESIGN: Eight male participants consumed 500 mg isotopically labeled cyanidin-3-glucoside (6,8,10,3',5'-(13)C5-C3G). Biological samples were collected over 48 h, and (13)C and (13)C-labeled metabolite concentrations were measured by using isotope-ratio mass spectrometry and liquid chromatography-tandem mass spectrometry. RESULTS: The mean ± SE percentage of (13)C recovered in urine, breath, and feces was 43.9 ± 25.9% (range: 15.1-99.3% across participants). The relative bioavailability was 12.38 ± 1.38% (5.37 ± 0.67% excreted in urine and 6.91 ± 1.59% in breath). Maximum rates of (13)C elimination were achieved 30 min after ingestion (32.53 ± 14.24 µg(13)C/h), whereas (13)C-labeled metabolites peaked (maximum serum concentration: 5.97 ± 2.14 µmol/L) at 10.25 ± 4.14 h. The half-life for (13)C-labeled metabolites ranged between 12.44 ± 4.22 and 51.62 ± 22.55 h. (13)C elimination was greatest between 0 and 1 h for urine (90.30 ± 15.28 µg/h), at 6 h for breath (132.87 ± 32.23 µg/h), and between 6 and 24 h for feces (557.28 ± 247.88 µg/h), whereas the highest concentrations of (13)C-labeled metabolites were identified in urine (10.77 ± 4.52 µmol/L) and fecal samples (43.16 ± 18.00 µmol/L) collected between 6 and 24 h. Metabolites were identified as degradation products, phenolic, hippuric, phenylacetic, and phenylpropenoic acids. CONCLUSION: Anthocyanins are more bioavailable than previously perceived, and their metabolites are present in the circulation for ≤48 h after ingestion. This trial was registered at clinicaltrials.gov as NCT01106729.

A new class of NO-donor pro-drugs triggered by γ-glutamyl transpeptidase with potential for reno-selective vasodilatation.

This communication describes the synthesis of a new class of N-hydroxyguanidine (NHG) pro-drugs which release nitric oxide (NO), triggered by the action of γ-glutamyl transpeptidase (γ-GT), and have potential for the treatment of acute renal injury/failure (ARI/ARF).

A gram scale synthesis of a multi-13C-labelled anthocyanin, [6,8,10,3',5'-13C5]cyanidin-3-glucoside, for use in oral tracer studies in humans.

The major dietary anthocyanin, cyanidin-3-glucoside, was prepared on a 4 g scale from three units of diethyl [2-(13)C]malonate and one unit of [1,3-(13)C(2)]acetone, such that five isotope locations were distributed throughout the molecule to provide a penta-(13)C(5)-labelled anthocyanin, [6,8,10,3',5'-(13)C(5)]cyanidin-3-glucoside chloride, for use in human stable-isotope tracer studies.

Bioavailability of Sulforaphane from two broccoli sprout beverages: results of a short-term, cross-over clinical trial in Qidong, China.

One of several challenges in design of clinical chemoprevention trials is the selection of the dose, formulation, and dose schedule of the intervention agent. Therefore, a cross-over clinical trial was undertaken to compare the bioavailability and tolerability of sulforaphane from two of broccoli sprout-derived beverages: one glucoraphanin-rich (GRR) and the other sulforaphane-rich (SFR). Sulforaphane was generated from glucoraphanin contained in GRR by gut microflora or formed by treatment of GRR with myrosinase from daikon (Raphanus sativus) sprouts to provide SFR. Fifty healthy, eligible participants were requested to refrain from crucifer consumption and randomized into two treatment arms. The study design was as follows: 5-day run-in period, 7-day administration of beverages, 5-day washout period, and 7-day administration of the opposite intervention. Isotope dilution mass spectrometry was used to measure levels of glucoraphanin, sulforaphane, and sulforaphane thiol conjugates in urine samples collected daily throughout the study. Bioavailability, as measured by urinary excretion of sulforaphane and its metabolites (in approximately 12-hour collections after dosing), was substantially greater with the SFR (mean = 70%) than with GRR (mean = 5%) beverages. Interindividual variability in excretion was considerably lower with SFR than with GRR beverage. Elimination rates were considerably slower with GRR, allowing for achievement of steady-state dosing as opposed to bolus dosing with SFR. Optimal dosing formulations in future studies should consider blends of sulforaphane and glucoraphanin as SFR and GRR mixtures to achieve peak concentrations for activation of some targets and prolonged inhibition of others implicated in the protective actions of sulforaphane. Cancer Prev Res; 4(3); 384-95. ©2011 AACR.

Flavonoid conjugates interact with organic anion transporters (OATs) and attenuate cytotoxicity of adefovir mediated by organic anion transporter 1 (OAT1/SLC22A6).

Flavonoids are conjugated by phase II enzymes in humans to form glucuronidated and sulfated metabolites that are excreted in urine via the kidney. In this study, we examined the interaction between metabolites of quercetin and isoflavonoids found in vivo with human organic anion transporters 1 (OAT1) and 3 (OAT3) and their potential in attenuating OAT-induced cytotoxicity of adefovir. Accumulation of flavonoid conjugates was studied in human embryonic kidney 293H cells overexpressing OAT1 or OAT3. OAT1-overexpressing cells exhibited an increased uptake of the sulfated conjugates, genistein-4'-O-sulfate and quercetin-3'-O-sulfate. OAT3-overexpressing cells demonstrated enhanced uptake of glucuronide conjugates, such as daidzein-7-O-glucuronide, genistein-7-O-glucuronide, glycitein-7-O-glucuronide and quercetin-3'-O-glucuronide. Position of conjugation was important since quercetin-3-O-glucuronide and quercetin-7-O-glucuronide were poorly transported. Kinetic analysis revealed high affinity uptake of quercetin-3'-O-sulfate by OAT1 (K(m)=1.73µM; V(max)=105 pmol/min/mg). OAT3 transported isoflavone glucuronides with lower affinity (K(m)=7.9-19.1 µM) but with higher V(max) (171-420 pmol/min/mg). Quercetin-3'-O-sulfate strongly inhibited OAT1-mediated p-aminohippuric acid uptake with an IC(50) of 1.22µM. Transport of 5-carboxyfluorescein by OAT3 was potently inhibited by quercetin-3-O-glucuronide, quercetin-3'-O-glucuronide and quercetin-3'-O-sulfate (IC(50)=0.43-1.31µM). In addition, quercetin-3'-O-sulfate was shown to effectively reduce OAT1-mediated cytotoxicity of adefovir, an antiviral drug, in a dose-dependent manner. These data suggest that OAT1 and OAT3 are responsible for basolateral uptake of flavonoid conjugates in kidney, and flavonoid conjugates inhibit OAT1 and OAT3 activity at physiologically relevant concentrations. Interaction with OATs limits systemic availability of flavonoids and may be a mechanism of food-drug interaction via inhibition of renal uptake.

First synthesis of [1,3,5-(13)C3]gallic acid.

An efficient and high-yielding synthesis for [1,3,5-(13)C(3)]gallic acid from non-aromatic precursors is presented. [3,5-(13)C(2)]4H-Pyran-4-one was first prepared from the reaction between triethyl orthoformate and [1,3-(13)C(2)]acetone. The third (13)C-atom was introduced into the ring by reaction of the pyranone with diethyl [2-(13)C]malonate. The resulting ethyl 4-hydroxy-[1,3,5-(13)C(3)]benzoate was brominated in the 3- and 5-positions to give ethyl 3,5-dibromo-4-hydroxy-[1,3,5-(13)C(3)]benzoate. Subsequent hydrolysis of the ester and substitution of the bromine atoms with hydroxyl groups was achieved under basic conditions in a single step to yield the desired [1,3,5-(13)C(3)]gallic acid. The synthesis of [2,6-(13)C(2)]4H-pyran-4-one is also presented to demonstrate the potential of the methodology for the regioselective placement of (13)C-atoms into benzene rings.

Orally administered isoflavones are present as glucuronides in the human prostate.

Better knowledge of the bioavailability and metabolism of isoflavones in prostate tissue is needed to further investigate their mechanisms of action in the context of prostate cancer prevention. A total of 12 men with benign prostatic hyperplasia received soy extract supplementation (3 Evestrel capsules, providing a total of 112.5 mg isoflavones aglycone eq/day) for 3 days before prostate surgery. Blood and prostate tissues were sampled and metabolites were identified using electrospray ionization liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS) and chemically synthesized standards of glucuronidated isoflavones. The main metabolites were the same in prostate tissue and in plasma, namely, 2 monoglucuronides of daidzein and 2 monoglucuronides of genistein. Concentrations of total isoflavones measured in prostate reached 1.05 +/- 0.62 nmol/g tissue (range 0.30-2.23) at the time of sampling, 12 h after the last isoflavone supplementation. At that time point, prostate concentrations were lower than plasma concentrations in all volunteers: 0.47 nmol/g vs. 0.66 microM for daidzein and 0.58 nmol/g vs. 0.78 microM for genistein. Isoflavone mechanisms of action should thus be investigated in in vitro cell studies using physiological conditions, intracellular concentrations below 5 nmol/g and no intracellular deconjugation of the monoglucuronide metabolites.

1-(5-Chloro-2,4-dihydroxy-phen-yl)-2-(4-ethoxy-phen-yl)ethanone.

The structure of the title compound, C(16)H(15)ClO(4), contains aryl rings which are inclined by 75.6 (1)° to each other. It displays intra-molecular O-H⋯O hydrogen bonding between the 2-hydr-oxy and carbonyl groups, forming a six-membered ring. Furthermore, the 4-hydr-oxy group, acting as a hydrogen-bond donor, is bound to the O atom of the 2-hydr-oxy group of another mol-ecule.

Synthesis of multiply 13C-labeled furofuran lignans using 13C-labeled cinnamyl alcohols as building blocks.

Plant lignans are currently being widely studied for their potential benefits for human health as their consumption has been correlated with lower risks for developing chronic diseases, such as breast cancer and coronary heart disease. However, studies of some classes of lignans, in particular the furofurans, are hampered by the lack of suitable standards to allow accurate analysis. Herein, we report the syntheses of two racemic (13)C-labeled furofuran lignans [7,8,9-(13)C(3)]medioresinol and [7,8,9-(13)C(3)]sesamin as internal standards for LC-MS analysis. The labeled furofuran lignans were constructed from triply labeled cinnamyl alcohols, using a radical cyclization method.

Analysis of glucosinolates, isothiocyanates, and amine degradation products in vegetable extracts and blood plasma by LC-MS/MS.

Dietary glucosinolates are under intensive investigation as precursors of cancer-preventive isothiocyanates. Quantitation of the dose and bioavailability of glucosinolates and isothiocyanates requires a comprehensive analysis of the major dietary glucosinolates, isothiocyanates, and related metabolites. We report a liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) analytical method for the comprehensive analysis of the seven major dietary glucosinolates, related isothiocyanates, and putative amine degradation products. The parent glucosinolates were sinigrin, gluconapin, progoitrin, glucoiberin, glucoraphanin, glucoalyssin, and gluconasturtiin. The LC-MS/MS analysis method for these compounds was developed and validated; a standard addition analysis protocol was used generally to avoid the requirement for stable isotopic standards. Where stable isotopic standards were available, internal standardization with these gave estimates in agreement with those obtained by the standard addition analysis protocol. For glucosinolates, negative ion electrospray LC-MS/MS analysis was performed. Isothiocyanates and amines were prederivatized to the corresponding thiourea and N-acetamides, respectively, and were quantified by positive ion electrospray LC-MS/MS. The limits of detection were 0.5-2 pmol; the recoveries for glucosinolates, isothiocyanates, and amines were 85-90%, 50-85%, and 60-70%, respectively; and the intra- and interbatch coefficients of variation were 1-4% and 3-10%, respectively. These methods provide facile access to comprehensive analytical data on the major dietary glucosinolates and related metabolites to quantify inputs and metabolic formation of these compounds in cancer prevention and related studies.

Metabolism of phytoestrogen conjugates.

Phytoestrogens are plant-derived compounds with physiologic estrogenic effects. They are present in the plant as glycosidic conjugates, some of which contain further chemical modifications (acetate, malonate, and 3-hydroxy-3-methylglutarate esters and 2,3-dihydroxysuccinate ether). In the gastrointestinal tract, the conjugates undergo hydrolysis catalyzed by enzymes in the intestinal wall and by gut bacteria. On entering the systemic circulation, the phytoestrogens may undergo extensive metabolism to other compounds through reactions involving demethylation, methylation, hydroxylation, chlorination, iodination, and nitration. In addition, all these compounds can undergo conjugation to form beta-glucuronides and sulfate esters. This chapter describes the methods of analysis of all these compounds, the sources of or methods to manufacture suitable standards, and the procedures for examining the enzymes that catalyze these reactions.

Phytoestrogen concentrations in serum and spot urine as biomarkers for dietary phytoestrogen intake and their relation to breast cancer risk in European prospective investigation of cancer and nutrition-norfolk.

Subjects of this study consisted of 333 women (aged 45-75 years) drawn from a large United Kingdom prospective study of diet and cancer, the European Prospective Investigation of Cancer and Nutrition-Norfolk study. Using newly developed gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry methods incorporating triply (13)C-labeled standards, seven phytoestrogens (daidzein, genistein, glycitein, O-desmethylangolensin, equol, enterodiol, and enterolactone) were measured in 114 spot urines and 97 available serum samples from women who later developed breast cancer. Results were compared with those from 219 urines and 187 serum samples from healthy controls matched by age and date of recruitment. Dietary levels were low, but even so, mean serum levels of phytoestrogens were up to 600 times greater than postmenopausal estradiol levels. Phytoestrogen concentrations in spot urine (adjusted for urinary creatinine) correlated strongly with that in serum, with Pearson correlation coefficients > 0.8. There were significant relationships (P < 0.02) between both urinary and serum concentrations of isoflavones across increasing tertiles of dietary intakes. Urinary enterodiol and enterolactone and serum enterolactone were significantly correlated with dietary fiber intake (r = 0.13-0.29). Exposure to all isoflavones was associated with increased breast cancer risk, significantly so for equol and daidzein. For a doubling of levels, odds ratios increased by 20-45% [log(2) odds ratio = 1.34 (1.06-1.70; P = 0.013) for urine equol, 1.46 (1.05-2.02; P = 0.024) for serum equol, and 1.22 (1.01-1.48; P = 0.044) for serum daidzein]. These estimates of risk are similar to those established for estrogens and androgens in postmenopausal breast cancer but need confirmation in larger studies.

Neutrophil myeloperoxidase chlorinates and nitrates soy isoflavones and enhances their antioxidant properties.

Soy isoflavones and other polyphenolics have a number of potentially important beneficial effects on the pro-oxidant aspects of chronic inflammation. The impact of inflammatory cell-specific metabolism of polyphenolics, which can include halogenation and nitration, on the properties of these compounds has not been examined. Using either human neutrophils or differentiated human leukemia cells (HL-60) stimulated with phorbol ester to elicit a respiratory burst, the hypothesis that local generation of reactive oxygen and nitrogen species may metabolize and modify the biological properties of the soy isoflavones was examined. Coincubation of the stimulated cells with genistein or daidzein had no effect on the respiratory burst. Medium from stimulated cells in the presence of the isoflavones and NO(2)(-) increased the inhibition of copper-induced LDL oxidation. Mass spectrometry analysis of this medium revealed that monochlorinated, dichlorinated, and nitrated isoflavones, formed through a myeloperoxidase-dependent mechanism, were present. The consumption of genistein in the presence of cells was both extensive and rapid with > 95% of the genistein converted to either the chlorinated or nitrated metabolites within 30 min. Chemically synthesized 3'-chlorogenistein and 3'-chlorodaidzein increased the inhibition of LDL oxidation by approximately 4-fold and 2-fold over genistein and daidzein, respectively. These results lead to the hypothesis that inflammatory cell-specific metabolism of polyphenolics can modify the properties of these compounds at the local site of inflammation.

Comparative inhibition by substrate analogues 3-methoxy- and 3-hydroxydesaminokynurenine and an improved 3 step purification of recombinant human kynureninase.

BACKGROUND: Kynureninase is a key enzyme on the kynurenine pathway of tryptophan metabolism. One of the end products of the pathway is the neurotoxin quinolinic acid which appears to be responsible for neuronal cell death in a number of important neurological diseases. This makes kynureninase a possible therapeutic target for diseases such as Huntington's, Alzheimer's and AIDS related dementia, and the development of potent inhibitors an important research aim. RESULTS: Two new kynurenine analogues, 3-hydroxydesaminokynurenine and 3-methoxydesaminokynurenine, were synthesised as inhibitors of kynureninase and tested on the tryptophan-induced bacterial enzyme from Pseudomonas fluorescens, the recombinant human enzyme and the rat hepatic enzyme. They were found to be mixed inhibitors of all three enzymes displaying both competitive and non competitive inhibition. The 3-hydroxy derivative gave low Ki values of 5, 40 and 100 nM respectively. An improved 3-step purification scheme for recombinant human kynureninase was also developed. CONCLUSION: For kynureninase from all three species the 2-amino group was found to be crucial for activity whilst the 3-hydroxyl group played a fundamental role in binding at the active site presumably via hydrogen bonding. The potency of the various inhibitors was found to be species specific. The 3-hydroxylated inhibitor had a greater affinity for the human enzyme, consistent with its specificity for 3-hydroxykynurenine as substrate, whilst the methoxylated version yielded no significant difference between bacterial and human kynureninase. The modified purification described is relatively quick, simple and cost effective.

Quantification of isoflavones and lignans in serum using isotope dilution liquid chromatography/tandem mass spectrometry.

Phytoestrogens (isoflavones and lignans) are receiving increasing attention due to a potential protective effect against a number of complex diseases. However, in order to investigate these associations, it is necessary to accurately quantify the levels of phytoestrogens in foods and biological fluids. We report an assay for three isoflavones (daidzein, genistein, and glycitein), two metabolites of daidzein (O-desmethylangolensin and equol), and two lignans (enterodiol and enterolactone) in human serum using electrospray ionisation liquid chromatography/mass spectrometry (LC/MS) with selective reaction monitoring. A simple, highly automated sample preparation procedure requires only 200 microL of sample and utilises one solid-phase extraction stage. Limits of detection are in the region of 10 pg/mL for all analytes except equol, which had a limit of detection of approximately 100 pg/mL. The method developed is suitable for measuring the concentrations of phytoestrogens in blood samples collected from large epidemiological studies. The results of the analysis of serum samples from 300 men and women living in the UK are reported.